ABSTRACT
OBJECTIVE： To optimize the proteolytic enzymes for enzymolysis technology of degreasing ointment from Periplaneta americana， and to improve the extraction rate and activity of anti-liver fibrosis active part from P. americana. METHODS： Using degreasing ointment of P. americana as control， ninhydrin method and folin-ciocalteu method were used to investigate the hydrolysis degree of trypsin （TR）， pepsin （PE）， alkaline protease （AL）， papain （PA） and neutral protease （NE） to the degreasing ointment. Macroporous resin isolation and purification method was used to investigate the yield of elution part from hydrolyzate， with 50％， 60％， 70％， 95％ ethanol as eluting solvents. Inhibition test in vitro of rat hepatic stellate cells HSC-T6 was performed， and anti-liver fibrosis activity of elution part from hydrolyzate was investigated. RESULTS： The hydrolysis degree of PA and NE were 14.15％ and 15.70％， showing strong enzymatic hydrolysis ability. The yield of 95％ ethanol elution part from PA， NE and AL hydrolyzate were （0.73±0.04）％，（0.65±0.01）％ and（0.64±0.05）％， improving 30.36％， 16.07％， 14.29％ compared with degreasing ointment without enzyme. Results of inhibition test in vitro showed that inhibitory rate of 50％， 60％， 70％ ethanol elution parts isolated and purified from hydrolyzate had a low inhibition rate or a growth-promoting effect on HSC-T6 cells. Inhibition rates of 95％ ethanol elution parts to HSC-T6 cells were all more than 20％. IC50 of 95％ ethanol elution part isolated and purified from PA and NE hydrolyzate for 24-72 h were 94.5-112.3 and 117.1-120.0 μg/mL， which were lower than that （116.1-123.0 μg/mL） of degreasing ointment without enzyme. CONCLUSIONS： PA is the best hydrolyzate for enzymolysis technology of active parts against liver fibrosis in degreasing ointment from P. americana， followed by NE and AL； PE and TR， which have poor effect， are not suitable for the enzymatic hydrolysis technology.